This proposal describes molecular studies on the structure and function of the serine protease (NS2B-Ns3 protease) of yellow fever virus. This virus is the prototype member of the flavivirus family which includes a large number of arthropod-transmitted human pathogens causing epidemic disease on a worldwide scale. The major objectives of the proposal are to advance understanding of the molecular basis for the formation and function of the viral NS2B-NS3 protease complex and to study its the role in regulating polyprotein processing during viral replication in cell culture. A related objective is to determine whether the activity of the protease can be inhibited using noncleavable or poorly-cleavable substrates. Information from these areas of investigation may be applicable to the development of antiviral agents which target this key viral protein. Such agents might have a role in the control of diseases caused by flaviviruses and provide insight into antiviral strategies against related viruses such as the pestiviruses and the hepatitis C viruses. The first specific aim is to determine if the critical amino acid residue within the NS2B and NS3 proteins required for formation of the protease complex can be defined using site-directed mutagenesis. This will be achieved by engineering a complete series of charged-to-alanin mutations within a NS2B-NS3 (181) polyprotein. The effects of such mutations on cleavage activity of the protease and formation of the complex will be studied using cell-free and cellular transient expression systems designed to assess cis and trans cleavage activities. The second aim is to determine whether selected mutations affect viral replication, and in particular whether conditional phenotypes in proteases function can be demonstrated. This will be achieved by introducing mutations into a molecular clone of the YF17D virus and evaluating growth properties and temperature sensitivity of viable clones. The third aim is to use a set of previously derived viral cleavage site mutants which exhibit impaired growth in cell to determine if specific defects in viral polyprotein processing can be observed and to determine how these are related to viral RNA synthesis. The fourth aim is to use a cell-free trans cleavage assay to determine if substrates containing mutations which block or reduce efficiency of cleavage at a native site within the nonstructural polyprotein can function as protease inhibitors.